In order to understand the development and functioning of the thymus, both in terms of T cell differentiation and stromal cell environmental support, we have undertaken a molecular approach to identify genes that are uniquely expressed in this organ. We have created a RT-PCR based subtracted cDNA library from fetal thymic stromal cells. The novel cDNAs were then screened for expression in various tissues and in a set of SV40 transformed thymic epithelial cell lines. Four genes were selected for further analysis because they were limited in their expression to the thymus or to one of the thymic stromal cell lines. All four were sequenced by obtaining full length cDNA clones from a SCID thymus library. The three novel genes were named Epithin, Spatial, and Thymic Stroma Co-Transporter (TSCOT). During the past year the lab has made progress in understanding the structure and function of three of these genes and their encoded proteins. 1) We characterized the human and mouse genomic structures at the TSCOT locus (now designated as ly110) and showed that there are evolutionarily conserved potential tissue-specific gene regulatory sequences in the region upstream of the transcription start site. 2) The organization of the mouse Laminin 5 locus is very similar to that of the previously described human locus; however, alternative messages in the two species show completely different usage of alternative transcriptional initiation and splicing mechanisms. 3). Epithin, a type II membrane serine protease, is rapidly processed in the cell during de novo biosynthesis and then localized in the plasma membrane. Two fragments of the processed protein (the N-terminus, containing the transmembrane region, and the extra cellular C-terminus, containing the serine protease domain, are linked by a noncovalent association in the plasma membrane. Later the serine protease domain is secreted into the medium. 4), A transgenic mouse line containing 3.1kb of the TSCOT promoter driving Enhanced Green Fluorescent Protein (EGFP) was established to identify the thymic stromal cell lineage in which TSCOT is expressed. Surprisingly, this mouse showed EGFP expression in the subcapsular and medullary compartments of the thymus in contrast to the cortex where the endogenous gene is expressed. Using this information, we are now attempting to generate mouse lines with ablations in these specific thymic stromal cell compartments by transgenic and knock-in targeting approaches.